Date Published: February 21, 2018
Publisher: Public Library of Science
Author(s): Liisa T. Chisty, Daniela Quaglia, Martin R. Webb, Maria Spies.
Single-stranded DNA (ssDNA) is a product of many cellular processes that involve double-stranded DNA, for example during DNA replication and repair, and is formed transiently in many others. Measurement of ssDNA formation is fundamental for understanding many such processes. The availability of a fluorescent biosensor for the determination of single-stranded DNA provides an important route to achieve this. Single-stranded DNA binding proteins (SSBs) protect ssDNA from degradation, but can be displaced to allow processing of the ssDNA. Their tight binding of ssDNA means that they are very good candidates for the development of a biosensor. Previously, the single stranded DNA binding protein from Escherichia coli, labeled with a fluorophore, (DCC-EcSSB) was developed and used for this purpose. However, the multiple binding modes of this protein meant that interpretation of DCC-EcSSB fluorescence was potentially complex in terms of determining the amount of ssDNA. Here, we present an improved biosensor, developed using the tetrameric SSB from Plasmodium falciparum as a new scaffold for fluorophore attachment. Each subunit of this tetrameric SSB was labeled with a diethylaminocoumarin fluorophore at a single site on its surface, such that there is a very large, 20-fold, fluorescence increase when it binds to ssDNA. This adduct can be used as a biosensor to report ssDNA formation. Because SSB from this organism has a single mode of binding ssDNA, namely 65–70 bases per tetramer, over a wide range of conditions, the fluorescent SSB allows simple quantitation of ssDNA. The binding is fast, possibly diffusion controlled, and tight (dissociation constant for DCC-PfSSB <5 pM). Its suitability for real-time assays of ssDNA formation was demonstrated by measurement of AddAB helicase activity, unwinding double-stranded DNA.
Many cellular processes involving DNA, such as replication, transcription and repair rely on separating double-stranded DNA (dsDNA) to form single-stranded DNA (ssDNA). Measurement of ssDNA formation is fundamental for understanding many such processes, in particular the activity of the large number of helicases that catalyze the strand separation using the energy of ATP hydrolysis. DNA helicase activity can be assayed directly by measuring either the formation ssDNA or loss of dsDNA. Several assays have been developed, mostly based on fluorescence of intercalating dyes, whereby the dye binds to dsDNA preferentially over ssDNA [1, 2] and bound dye is depleted as ssDNA forms. However, such depletion assays modify the substrate, here dsDNA, and so may alter the activity being measured. It is often preferred to measure the product ssDNA, as the measurement is less likely to interfere with the activity.